The dentate gyrus generates new neurons throughout life, long after the neurogenesis of early development has ended. Adult neurogenesis makes important contributions to neuroplasticity and learning, and its impairment has been linked to neurodegeneration, learning disability, and epilepsy. New adult-born neurons integrate into existing mature neural circuitry, but retain the physiological attributes of immature neurons. Compared to mature neurons, new adult-born neurons form synapses more readily, are more excitable, and are more plastic with respect to induction of long-term potentiation of synaptic transmission. The study of adult neurogenesis is a very active field, and dramatic advances are being made in understanding the molecular control mechanisms and cellular properties of new adult-born neurons. Although the circuitry and network properties of new adult-born neurons ultimately play a critical role in their functions, the lack of suitable experimental tools and methods has impeded progress in this direction. This proposal will investigate network activity of new adult-born neurons by using a genetically targetable fluorescent probe in the hybrid voltage sensor (hVOS) family. hVOS employs a fluorescent protein that can be targeted for expression in defined populations of cells. hVOS imaging can record action potentials and subthreshold synaptic potentials from single neurons in hippocampal slices in a single trial (without averaging). We propose to express hVOS probe in newborn neurons and image their electrical activity. We will express probe in newborn neurons with retrovirus, as well as with a new hVOS Cre reporter mouse that can target probe expression to genetically defined populations of cells by crossing with Cre drivers. We will use an inducible Tbr2-CreERT2 driver to target probe to new adult-born neurons with high temporal resolution. The two expression approaches will be compared to check for consistency. hVOS imaging in hippocampal slices will then allow us to monitor the electrical activity of multiple new adult-born neurons simultaneously. We will stimulate the different granule cell inputs, perforant path axons and mossy cells, and use hVOS to record subthreshold synaptic potentials and action potentials. Experiments will test hypotheses about the organization of newborn neuron circuits by evaluating the properties, correlations, and synchronization of evoked electrical responses recorded in many new adult-born neurons simultaneously. We will directly test the hypothesis that mature granule cells synapse with immature granule cells. We will use this imaging approach to determine whether new neurons integrate into existing neural circuitry as functional clusters with shared synaptic inputs, evaluate clones of newborn neurons descended from a common progenitor cell, and explore the evolution of circuits as neurons mature. This work will reveal the circuit relations of new adult-born neurons, and will introduce a novel technique for the study of neural networks to the field of adult neurogenesis.